A doxorubicin-CNGRC-peptide conjugate with prodrug properties

There is increasing interest in the exploitation of molecular addresses for the targeting of tumor imaging or therapeutic agents. A recent study demonstrated anticancer activity in human xenografts of doxorubicin (DOX)-peptide conjugates targeted to the tumor vascular endothelium, among them DOX coupled to the cyclic pentapeptide CNGRC [Science 279 (1998) 377]. In order to learn more about the mechanism of action of this type of DOX-peptide conjugates, we have studied the interaction of DOX-CNGRC with primary human umbilical cord vein endothelial cells (HUVEC) and tumor cells under defined in vitro conditions. We used a DOX conjugate, in which the cyclic CNGRC peptide, for which an in vivo endothelial address has recently been identified as aminopeptidase N (APN)/CD13, has been coupled via a hydrolysable spacer to the C-14 anthracycline-side chain. First we determined that the t(1/2) of DOX-CNGRC conjugate in human blood was 442 min (at 37 degrees ) allowing sufficient time for endothelial targeting when administered i.v. When cultured cells were exposed for 30 min to DOX-CNGRC a more cytoplasmic localization of fluorescent drug was seen when compared to DOX exposure and intracellular DOX-CNGRC was identified after extraction from the cells. This revealed differences in the cellular uptake process of the conjugate compared to DOX. The antiproliferative effect of DOX-CNGRC was determined by 30 min exposure in medium with a high protein content in order to mimick the in vivo targeting situation. In this medium, the IC(50) was 1.1 microM for highly CD13 expressing HT-1080, 1.45 microM for CD13 negative SK-UT-1 sarcoma cells and 6.5 microM for CD13 positive HUVEC. The IC(50) of DOX for these cells were 1.0, 2.0 and 7.3 microM, respectively. Although DOX-CNGRC inhibited the peptidase activity of CD13 up to 50%, our data do not favor an important role for the enzyme inhibition in the cytotoxic effect of the conjugate. The antitumor activity was tested in nude mice bearing human ovarian cancer xenografts (OVCAR-3). A weekly i.v. administration (3mg/kg DOX-equivalent, 3x) showed a minor (40%) growth delay, which does not indicate efficacy better than that expected for free DOX. In conclusion, this study indicates that the antiproliferative and anti-angiogenic effects of DOX-CNGRC as reported before, are likely caused by the cytostatic effects of intracellularly released parent drug DOX, independent of CD13 expression/activity. More research is needed to identify the optimal specific chemical configuration of DOX-peptide conjugates for in vivo targeting and receptor-mediated cellular uptake.     

Triggering endogenous immunosuppressive mechanisms by combined targeting of Dipeptidyl peptidase IV (DPIV/CD26) and Aminopeptidase N (APN/ CD13) — A novel approach for the treatment of inflammatory bowel disease

The ectopeptidases Dipeptidylpeptidase IV and Alanyl-Aminopeptidase N, strongly expressed by both, activated and regulatory T cells were shown to co-operate in T cell regulation. Based on the findings that DPIV and APN inhibitors induce the TGF-β1 and IL-10 production and a suppression of T helper cell proliferation in parallel, and that particularly APN inhibitors amplify the suppressing activity of regulatory T cells, both peptidases represent a promising target complex for treatment of diseases associated with an imbalanced T cell response, such as inflammatory bowel diseases (IBD).The aim of the present study was to analyze the therapeutic potential of DPIV and APN inhibitors in vivo in a mouse model of colitis. Balb/c mice received 3% (w/v) dextran sulphate sodium with the drinking water for 7 days. After onset of colitis symptoms, inhibitor treatment started at day 3. Disease activity index (DAI) was assessed daily, supplemented by histological and immunological analysis. While the DPIV inhibitor Lys-[Z(NO])₂]-pyrrolidide or the APN-inhibitor Actinonin alone had marked but no significant therapeutic effects, the simultaneous administration of both inhibitors reduced colitis activity in comparison to placebo treated mice, significantly (DAI 4.8 vs. 7.7, p < 0.005). A newly developed compound IP12.C6 with inhibitory capacity toward both enzymes significantly attenuated the clinical manifestation of colitis (DAI 3.2 vs. 7.6, p < 0.0001). TGF-β mRNA was found to be up-regulated in colon tissue of inhibitor-treated animals.In summary our results strongly suggest that combined DPIV and APN inhibition by synthetic inhibitors represents a novel and efficient approach for the pharmacological therapy of IBD by triggering endogenous immunosuppressive mechanisms.     

左卡尼汀联合前列地尔对冠心病合并2型糖尿病患者血液流变学、IMT及血清APN、Hcy的影响

目的观察左卡尼汀联合前列地尔对冠心病合并2型糖尿病患者血液流变学、IMT及血清APN、Hcy的影响。方法选取甘肃省平凉和平医院2015年4月—2016年10月收治的冠心病合并2型糖尿病患者106例,将其分为研究组和对照组,每组各53例。对照组在常规治疗基础上给予前列地尔治疗,研究组在对照组基础上加用左卡尼汀治疗,观察两组治疗前后患者血液流变学、颈动脉内中膜厚度(Intima-media thickness,IMT)及血清脂联素(Adiponectin,APN)、同型半胱氨酸(Homocysteine,Hcy)水平变化情况,比较两组的总有效率和不良反应情况。结果治疗后两组患者血浆黏度、红细胞压积、红细胞变形指数和纤维蛋白原等血液流变学指标水平均比治疗前明显改善,且研究组优于对照组(P0.05);治疗后两组IMT厚度比治疗前无显著增加(P0.05);治疗后两组患者血清APN水平比治疗前显著升高(P0.05),Hcy水平均显著降低(P0.05),且研究组优于对照组(P0.05);研究组总有效率为94.34%,明显高于对照组的79.25%(P0.05);研究组不良反应率为13.21%,与对照组11.32%无明显差异(P0.05)。结论左卡尼汀联合前列地尔治疗冠心病合并2型糖尿病,能改善血液流变学指标,稳定患者IMT厚度,提高血清APN水平,降低血清Hcy水平。     

中西医结合治疗急性脑梗死的临床疗效及对血vWF、APN水平的影响

目的探讨中西医结合疗法对临床急性脑梗死(ACI)患者的疗效及对血管性血友病因子(vWF)、血清脂联素(APN)水平的影响。方法选取2013年6月至2014年6月本院收治的ACI患者96例,依据随机数字表法分为研究组与对照组各48例。对照组予以西医常规治疗,研究组另给予中药汤剂治疗,比较治疗2周后两组的疗效,治疗前后的血vWF、APN水平及神经功能缺损(NIHSS)评分,分析NIHSS评分与血vWF、APN水平的相关性。结果研究组的总有效率93.75%(45/48)明显高于对照组77.08%(37/48),差异有统计学意义(P0.05)。治疗后研究组的血vWF水平明显低于对照组,APN水平明显高于对照组,差异均有统计学意义(P0.05)。两组治疗后NIHSS评分均有改善,但研究组明显低于对照组,差异有统计学意义(P0.05)。依照Pearson相关分析可知,NIHSS评分与血vWF呈正相关,与APN呈负相关。结论中西医结合治法对ACI患者具有较好的疗效,还可有效改善患者的血vWF、APN指标水平,值得临床推广。     

CD13/APN生物学功能的研究进展

CD13／APN不仅是髓细胞表面标志,也分布于其它多种体细胞,具有重要的生物学功能。近年来的研究主要集中在其促血细胞分化发育、参与血管发生、调节免疫应答以及作为细胞表面受体发挥作用等方面。虽然其中很多环节的分子机制还有待进一步阐明,但CD13／APN已经显示出应用于科研和临床治疗的良好的潜力。     

Dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) as regulators of T cell function and targets of immunotherapy in CNS inflammation

The ectoenzymes dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) have been implicated in the regulation of T cell activation and function. Both DP IV and APN serve as targets of efficient enzymatic inhibitors which induce autocrine production of TGF-beta1 and subsequent suppression of T cell proliferation and cytokine release. Here, we tested the hypothesis that the simultaneous inhibition of DP IV and APN enzymatic activity on leukocytes potentiates the anti-inflammatory effect of single DP IV or APN inhibitors. Our data show that the combined application of DP IV and APN inhibitors increased suppression of DNA synthesis in human peripheral blood mononuclear cells and isolated T cells in vitro when compared to the use of a single ectopeptidase inhibitor. Moreover, the combined action of DP IV and APN inhibitors markedly increased TGF-beta1 production associated with the observed immunosuppressive effects. In vivo, targeting DP IV and APN provided a potent therapeutic approach for the treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Taken together, our study suggests that combined DP IV and APN inhibition on pathogenic T cells represents a novel and efficient therapy for autoimmune disease of the central nervous system by a mechanism that involves an active TGF-beta1-mediated anti-inflammatory effect at the site of pathology.     

Aminopeptidase N (APN/CD13) is selectively expressed in vascular endothelial cells and plays multiple roles in angiogenesis

Proteolytic enzyme-mediated degradation of the extracellular matrix (ECM) is crucial for the formation of both tumor metastasis and angiogenesis. Recently, several reports have suggested that aminopeptidases are involved in this process, but precisely how is largely unknown. We found here that aminopeptidase N (APN/CD13) was selectively expressed in vascular endothelial cells including human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC), and was not detectable in a majority of normal cells and tumor cell lines we examined. RNA interference (RNAi) of APN resulted in the inhibition of capillary tube formation of HUVEC on Matrigel. APN siRNA suppressed the migration of HUVEC through a fibronectin-coated Transwell membrane, and reduced the cellular adhesion to Matrigel and various adhesion molecules including type IV collagen, type I collagen and fibronectin. These findings suggest that APN is a multifunctional protein with important roles in vascular endothelial morphogenesis during angiogenesis.     

冠脉通片联合氯吡格雷治疗冠心病心绞痛的临床研究

目的探讨冠脉通片联合硫酸氢氯吡格雷片治疗冠心病心绞痛的临床疗效。方法选取2019年5月—2020年5月在天津市南开区中医医院治疗的冠心病心绞痛患者94例,根据就诊顺序分为对照组(47例)和治疗组(47例)。对照组口服硫酸氢氯吡格雷片,75mg/次,1次/d;治疗组在对照组的基础上口服冠脉通片,1.8g/次,3次/d。两组患者均经4周治疗。观察两组患者心绞痛和心电图疗效,同时比较治疗前后两组患者心绞痛发作次数和持续时间,及血清血小板颗粒膜蛋白-140(GMP-140)、可溶性细胞间黏附分子-1(sICAM-1)、纤溶酶原激活剂抑制物-1(PAI-1)、白细胞三烯E4(LTB4)、髓过氧化物酶(MPO)、脂联素(APN)、血小板黏附率(PAR)、血浆黏度(PV)、纤维蛋白原(FIB)、全血黏度(WBV)、磷酸肌酸激酶(CK)、磷酸肌酸酶同工酶(CK-MB)、乳酸肌氢酶(LDH)和羟丁酸脱氢酶(HBDH)水平。结果治疗后,对照组心绞痛和心电图有效率分别为80.65%和82.98%,均分别显著低于治疗组的95.74%和97.87%,两组比较差异有统计学意义(P<0.05)。治疗后,两组心绞痛发作次数和持续时间均显著下降(P<0.05),且治疗组下降更明显(P<0.05)。治疗后,两组血清GMP-140、sICAM-1、PAI-1、LTB4、MPO、PV、WBV、PAR、FIB、CK、CK-MB、LDH、HBDH水平均显著下降(P<0.05),而APN显著升高(P<0.05),且治疗组这些血清学指标改善更明显(P<0.05)。结论冠心病心绞痛患者给予氯吡格雷治疗的同时口服冠脉通片可促进心绞痛症状改善,有利于心肌酶谱及血清GMP-140、sICAM-1、PAI-1、LTB4、MPO、APN水平改善,具有一定的临床推广应用价值。     

APN、hs-CRP、ET-1及NO在原发性高血压患者中的检测价值

目的 :探析血清脂联素(APN)、高敏C反应蛋白(hs-CRP)、内皮素1(ET-1)及一氧化氮(NO)在原发性高血压(PH)患者中的临床价值.方法 :分析2014年2月~2016年7月在我院接受诊治的98例PH患者的临床资料.另外选取我院同期接受健康体检的160例血压低于140/90mmHg者作为本次研究的对照组,并根据血压水平的高低分成正常血压组(NBP组,低于120/80mmHg,82例)和正常高值血压组(NHBP组,120~139/80~89mmHg,78例).比较三组患者的一般资料与血清APN、hs-CRP、ET-1及NO的表达水平,并相关性分析平均动脉压(MAP)、血清APN、hs-CRP、ET-1及NO,多因素Logistic回归分析PH患者MAP的影响因子.结果 :PH组患者的收缩压(SBP)、舒张压(DBP)及MAP水平显著较NBP组、NHBP组高,且NHBP组SBP、DBP及MAP水平又显著高于NBP组;三组患者其它一般资料(包括性别、年龄、BMI值等)无显著差异.PH组患者的血清hs-CRP、ET-1水平显著高于NBP组和NHBP组,而血清APN、NO水平则显著低于NBP组和NHBP组;与NBP组相比,NHBP组患者的血清APN及NP水平显著降低,血清hs-CRP与ET-1水平显著升高.血清APN、hs-CRP、ET-1、NO均是PH患者MAP的影响因子.结论 :PH患者体内血管舒缩及炎症因子平衡失调,主要表现为血清hs-CRP及ET-1水平上升,血清APN及NO水平降低,且这种失衡在NHBP患者中就已存在.     

扶正消瘀汤对糖尿病下肢动脉病变的血脂APN及血流状况的影响

目的:探讨扶正消瘀汤对糖尿病下肢动脉血管病变的血脂APN及血流状况的影响。方法:选取2015年3月至2016年3月荆门市第二人民医院收治的糖尿病下肢血管病变患者100例作为研究对象,按照患者入院ID号尾号随机分为对照组和观察组,每组50例。对照组采取常规治疗,观察组在对照组的基础上,采用扶正消瘀汤治疗,对2组患者治疗后的疗效、治疗前后的血液流变学及血清C反应蛋白(CRP)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、血脂脂联素(APN)、踝肱指数、足背动脉血流量以及不良反应进行比较分析。结果:治疗前,2组患者的血脂APN比较,差异无统计学意义(P 0. 05);治疗后,观察组的血脂APN明显高于对照组,2组比较差异有统计学意义(P 0. 05)。观察组患者的总有效率(94. 00%)明显小于对照组(80. 00%),治疗前,2组患者的全血低、高切黏度以及血浆黏度比较,差异无统计学意义(P 0. 05);治疗后,全血低、高切黏度以及血浆黏度明显低于对照组,CRP、IL-6、TNF-α水平均明显低于对照组,2组比较差异有统计学意义(P 0. 05)。治疗前,2组患者的踝肱指数和足背动脉血流量比较,差异无统计学意义(P 0. 05)。治疗后,观察组的踝肱指数和足背动脉血流量均明显高于对照组,2组比较差异有统计学意义(P 0. 05)。结论:对糖尿病下肢血管病变患者采取扶正消瘀汤治疗,能够提高临床疗效,改善患者血液流变学及血清水平,同时提高血脂APN水平,降低不良反应的发生率,值得临床上应用及推广。